Cohesin mediates DNA loop extrusion by a "swing and clamp" mechanism.

Structural maintenance of chromosomes (SMC) complexes organize genome topology in all kingdoms of life and have been proposed to perform this function by DNA loop extrusion. How this process works is unknown. Here, we have analyzed how loop extrusion is mediated by human cohesin-NIPBL complexes, which enable chromatin folding in interphase cells. We have identified DNA binding sites and large-scale conformational changes that are required for loop extrusion and have determined how these are coordinated. Our results suggest that DNA is translocated by a spontaneous 50 nm-swing of cohesin's hinge, which hands DNA over to the ATPase head of SMC3, where upon binding of ATP, DNA is clamped by NIPBL. During this process, NIPBL "jumps ship" from the hinge toward the SMC3 head and might thereby couple the spontaneous hinge swing to ATP-dependent DNA clamping. These results reveal mechanistic principles of how cohesin-NIPBL and possibly other SMC complexes mediate loop extrusion.

Identification of lectin receptors for conserved SARS-CoV-2 glycosylation sites.

New SARS-CoV-2 variants are continuously emerging with critical implications for therapies or vaccinations. The 22 N-glycan sites of Spike remain highly conserved among SARS-CoV-2 variants, opening an avenue for robust therapeutic intervention. Here we used a comprehensive library of mammalian carbohydrate-binding proteins (lectins) to probe critical sugar residues on the full-length trimeric Spike and the receptor binding domain (RBD) of SARS-CoV-2. Two lectins, Clec4g and CD209c, were identified to strongly bind to Spike. Clec4g and CD209c binding to Spike was dissected and visualized in real time and at single-molecule resolution using atomic force microscopy. 3D modelling showed that both lectins can bind to a glycan within the RBD-ACE2 interface and thus interferes with Spike binding to cell surfaces. Importantly, Clec4g and CD209c significantly reduced SARS-CoV-2 infections. These data report the first extensive map and 3D structural modelling of lectin-Spike interactions and uncovers candidate receptors involved in Spike binding and SARS-CoV-2 infections. The capacity of CLEC4G and mCD209c lectins to block SARS-CoV-2 viral entry holds promise for pan-variant therapeutic interventions.

Molecular Recognition in Confined Space Elucidated with DNA Nanopores and Single-Molecule Force Microscopy.

The binding of ligands to receptors within a nanoscale small space is relevant in biology, biosensing, and affinity filtration. Binding in confinement can be studied with biological systems but under the limitation that essential parameters cannot be easily controlled including receptor type and position within the confinement and its dimensions. Here we study molecular recognition with a synthetic confined nanopore with controllable pore dimension and molecular DNA receptors at different depth positions within the channel. Binding of a complementary DNA strand is studied at the single-molecule level with atomic force microscopy. Following the analysis, kinetic association rates are lower for receptors positioned deeper inside the pore lumen while dissociation is faster and requires less force. The phenomena are explained by the steric constraints on molecular interactions in confinement. Our study is the first to explore recognition in DNA nanostructures with atomic force microscopy and lays out new tools to further quantify the effect of nanoconfinement on molecular interactions.

Atomic Force Microscopy-Based Force Spectroscopy and Multiparametric Imaging of Biomolecular and Cellular Systems.

During the last three decades, a series of key technological improvements turned atomic force microscopy (AFM) into a nanoscopic laboratory to directly observe and chemically characterize molecular and cell biological systems under physiological conditions. Here, we review key technological improvements that have established AFM as an analytical tool to observe and quantify native biological systems from the micro- to the nanoscale. Native biological systems include living tissues, cells, and cellular components such as single or complexed proteins, nucleic acids, lipids, or sugars. We showcase the procedures to customize nanoscopic chemical laboratories by functionalizing AFM tips and outline the advantages and limitations in applying different AFM modes to chemically image, sense, and manipulate biosystems at (sub)nanometer spatial and millisecond temporal resolution. We further discuss theoretical approaches to extract the kinetic and thermodynamic parameters of specific biomolecular interactions detected by AFM for single bonds and extend the discussion to multiple bonds. Finally, we highlight the potential of combining AFM with optical microscopy and spectroscopy to address the full complexity of biological systems and to tackle fundamental challenges in life sciences.

Co-transient expression of PSA-Fc and PAP-Fc fusion protein in plant as prostate cancer vaccine candidates and immune responses in mice.

KEY MESSAGE: PAP-FcK and PSA-FcK prostate cancer antigenic proteins transiently co-expressed in plant induce their specific humoral immune responses in mice. Prostate-specific antigen (PSA) and prostatic acid phosphatase (PAP) have been considered as immunotherapeutic antigens for prostate cancer. The use of a single antigenic agent is unlikely to be effective in eliciting immunotherapeutic responses due to the heterogeneous and multifocal nature of prostate cancer. Thus, multiple antigens have been combined to enhance their anti-cancer effects. In the current study, PSA and PAP were fused to the crystallizable region (Fc region) of immunoglobulin G1 and tagged with KDEL, the endoplasmic reticulum (ER) retention signal motif, to generate PSA-FcK and PAP-FcK, respectively, and were transiently co-expressed in Nicotiana benthamiana. Western blot analysis confirmed the co-expression of PSA-FcK and PAP-FcK (PSA-FcK + PAP-FcK) with a 1:3 ratios in the co-infiltrated plants. PSA-FcK, PAP-FcK, and PSA-FcK + PAP-FcK proteins were successfully purified from N. benthamiana by protein A affinity chromatography. ELISA showed that anti-PAP and anti-PSA antibodies successfully detected PAP-FcK and PSA-FcK, respectively, and both detected PSA-FcK + PAP-FcK. Surface plasmon resonance (SPR) analysis confirmed the binding affinity of the plant-derived Fc fusion proteins to FcγRI/CD64. Furthermore, we also confirmed that mice injected with PSA-FcK + PAP-FcK produced both PSA- and PAP-specific IgGs, demonstrating their immunogenicity. This study suggested that the transient plant expression system can be applied to produce the dual-antigen Fc fusion protein (PSA-FcK + PAP-FcK) for prostate cancer immunotherapy.

Control of Ligand-Binding Specificity Using Photocleavable Linkers in AFM Force Spectroscopy.

In recent decades, atomic force microscopy (AFM), in particular the force spectroscopy mode, has become a method of choice to study biomolecular interactions at the single-molecule level. However, grafting procedures as well as determining binding specificity remain challenging. We report here an innovative approach based on a photocleavable group that enables in situ release of the ligands bound to the AFM tip and thus allows direct assessment of the binding specificity. Applicable to a wide variety of molecules, the strategy presented here provides new opportunities to study specific interactions and deliver single molecules with high spatiotemporal resolution in a wide range of applications, including AFM-based cell biology.

Simultaneous AFM topography and recognition imaging at the plasma membrane of mammalian cells.

Elucidation the nano-organization of membrane proteins at/within the plasma membrane is probably the most demanding and still challenging task in cell biology since requires experimental approaches with nanoscale resolution. During last decade, atomic force microscopy (AFM)-based simultaneous topography and recognition imaging (TREC) has become a powerful tool to quickly obtain local receptor nano-maps on complex heterogeneous biosurfaces such as cells and membranes. Here we emphasize the TREC technique and explain how to unravel the nano-landscape of mammalian cells. We describe the procedures for all steps of the experiment including tip functionalization with ligand molecules, sample preparation, and localization of key molecules on the cell surface. We also discuss the current limitations and future perspectives of this technique.

Localizing Binding Sites on Bioconjugated Hydrogen-Bonded Organic Semiconductors at the Nanoscale.

Hydrogen-bonded organic semiconductors are extraordinarily stable organic solids forming stable, large crystallites with the ability to preserve favorable electrical properties upon bioconjugation. Lately, tremendous efforts have been made to use these bioconjugated semiconductors as platforms for stable multifunctional bioelectronics devices, yet the detailed characterization of bio-active binding sites (orientation, density, etc.) at the nanoscale has not been achieved yet. The presented work investigates the bioconjugation of epindolidione and quinacridone, two representative semiconductors, with respect to their exposed amine-functionalities. Relying on the biotin-avidin lock-and-key system and applying the atomic force microscopy (AFM) derivative topography and recognition (TREC) imaging, we used activated biotin to flag crystal-faces with exposed amine functional groups. Contrary to previous studies, biotin bonds were found to be stable towards removal by autolysis. The resolution strength and clear recognition capability makes TREC-AFM a valuable tool in the investigation of bio-conjugated, hydrogen-bonded semiconductors.

Ultra-Sensitive and Label-Free Probing of Binding Affinity Using Recognition Imaging.

Reliable quantification of binding affinity is important in biotechnology and pharmacology and increasingly coupled with a demand for ultrasensitivity, nanoscale resolution, and minute sample amounts. Standard techniques are not able to meet these criteria. This study provides a new platform based on atomic force microscopy (AFM)-derived recognition imaging to determine affinity by visualizing single molecular bindings on nanosize dendrons. Using DNA hybridization as a demonstrator, an AFM sensor adorned with a cognate binding strand senses and localizes target DNAs at nanometer resolution. To overcome the limitations of speed and resolution, the AFM cantilever is sinusoidally oscillated close to resonance conditions at small amplitudes. The equilibrium dissociation constant of capturing DNA duplexes was obtained, yielding 2.4 × 10-10 M. Our label-free single-molecular biochemical analysis approach evidences the utility of recognition imaging and analysis in quantifying biomolecular interactions of just a few hundred molecules.

Unraveling the Macromolecular Pathways of IgG Oligomerization and Complement Activation on Antigenic Surfaces.

IgG antibodies play a central role in protection against pathogens by their ability to alert and activate the innate immune system. Here, we show that IgGs assemble into oligomers on antigenic surfaces through an ordered, Fc domain-mediated process that can be modulated by protein engineering. Using high-speed atomic force microscopy, we unraveled the molecular events of IgG oligomer formation on surfaces. IgG molecules were recruited from solution although assembly of monovalently binding molecules also occurred through lateral diffusion. Monomers were observed to assemble into hexamers with all intermediates detected, but in which only hexamers bound C1. Functional characterization of oligomers on cells also demonstrated that C1 binding to IgG hexamers was a prerequisite for maximal activation, whereas tetramers, trimers, and dimers were mostly inactive. We present a dynamic IgG oligomerization model, which provides a framework for exploiting the macromolecular assembly of IgGs on surfaces for tool, immunotherapy, and vaccine design.

Communication between N terminus and loop2 tunes Orai activation.

Ca2+ release-activated Ca2+ (CRAC) channels constitute the major Ca2+ entry pathway into the cell. They are fully reconstituted via intermembrane coupling of the Ca2+-selective Orai channel and the Ca2+-sensing protein STIM1. In addition to the Orai C terminus, the main coupling site for STIM1, the Orai N terminus is indispensable for Orai channel gating. Although the extended transmembrane Orai N-terminal region (Orai1 amino acids 73-91; Orai3 amino acids 48-65) is fully conserved in the Orai1 and Orai3 isoforms, Orai3 tolerates larger N-terminal truncations than Orai1 in retaining store-operated activation. In an attempt to uncover the reason for these isoform-specific structural requirements, we analyzed a series of Orai mutants and chimeras. We discovered that it was not the N termini, but the loop2 regions connecting TM2 and TM3 of Orai1 and Orai3 that featured distinct properties, which explained the different, isoform-specific behavior of Orai N-truncation mutants. Atomic force microscopy studies and MD simulations suggested that the remaining N-terminal portion in the non-functional Orai1 N-truncation mutants formed new, inhibitory interactions with the Orai1-loop2 regions, but not with Orai3-loop2. Such a loop2 swap restored activation of the N-truncation Orai1 mutants. To mimic interactions between the N terminus and loop2 in full-length Orai1 channels, we induced close proximity of the N terminus and loop2 via cysteine cross-linking, which actually caused significant inhibition of STIM1-mediated Orai currents. In aggregate, maintenance of Orai activation required not only the conserved N-terminal region but also permissive communication of the Orai N terminus and loop2 in an isoform-specific manner.

Lipoteichoic acid mediates binding of a Lactobacillus S-layer protein.

The Gram-positive lactic acid bacterium Lactobacillus buchneri CD034 is covered by a two-dimensional crystalline, glycoproteinaceous cell surface (S-) layer lattice. While lactobacilli are extensively exploited as cell surface display systems for applied purposes, questions about how they stick their cell wall together are remaining open. This also includes the identification of the S-layer cell wall ligand. In this study, lipoteichoic acid was isolated from the L. buchneri CD034 cell wall as a significant fraction of the bacterium's cell wall glycopolymers, structurally characterized and analyzed for its potential to mediate binding of the S-layer to the cell wall. Combined component analyses and 1D- and 2D-nuclear magnetic resonance spectroscopy (NMR) revealed the lipoteichoic acid to be composed of on average 31 glycerol-phosphate repeating units partially substituted with α-d-glucose, and with an α-d-Galp(1→2)-α-d-Glcp(1→3)-1,2-diacyl-sn-Gro glycolipid anchor. The specificity of binding between the L. buchneri CD034 S-layer protein and purified lipoteichoic acid as well as their interaction force of about 45 pN were obtained by single-molecule force spectroscopy; this value is in the range of typical ligand-receptor interactions. This study sheds light on a functional implication of Lactobacillus cell wall architecture by showing direct binding between lipoteichoic acid and the S-layer of L. buchneri CD034.

Inhibition of mitochondrial UCP1 and UCP3 by purine nucleotides and phosphate.

Mitochondrial membrane uncoupling protein 3 (UCP3) is not only expressed in skeletal muscle and heart, but also in brown adipose tissue (BAT) alongside UCP1, which facilitates a proton leak to support non-shivering thermogenesis. In contrast to UCP1, the transport function and molecular mechanism of UCP3 regulation are poorly investigated, although it is generally agreed upon that UCP3, analogous to UCP1, transports protons, is activated by free fatty acids (FFAs) and is inhibited by purine nucleotides (PNs). Because the presence of two similar uncoupling proteins in BAT is surprising, we hypothesized that UCP1 and UCP3 are differently regulated, which may lead to differences in their functions. By combining atomic force microscopy and electrophysiological measurements of recombinant proteins reconstituted in planar bilayer membranes, we compared the level of protein activity with the bond lifetimes between UCPs and PNs. Our data revealed that, in contrast to UCP1, UCP3 can be fully inhibited by all PNs and IC50 increases with a decrease in PN-phosphorylation. Experiments with mutant proteins demonstrated that the conserved arginines in the PN-binding pocket are involved in the inhibition of UCP1 and UCP3 to different extents. Fatty acids compete with all PNs bound to UCP1, but only with ATP bound to UCP3. We identified phosphate as a novel inhibitor of UCP3 and UCP1, which acts independently of PNs. The differences in molecular mechanisms of the inhibition between the highly homologous transporters UCP1 and UCP3 indicate that UCP3 has adapted to fulfill a different role and possibly another transport function in BAT.

Interaction of von Willebrand factor domains with collagen investigated by single molecule force spectroscopy.

von Willebrand factor (VWF) is a huge multimeric protein that plays a key role in primary hemostasis. Sites for collagen binding, an initial event of hemostasis, are located in the VWF-domains A1 and A3. In this study, we investigated single molecule interactions between collagen surfaces and wild type VWF A1A2A3 domain constructs, as well as clinically relevant VWF A3 domain point mutations, such as p.Ser1731Thr, p.Gln1734His, and p.His1786Arg. For this, we utilized atomic force microscopy based single molecular force spectroscopy. The p.Ser1731Thr mutant had no impact on the VWF-collagen type III and VI interactions, while the p.Gln1734His and p.His1786Arg mutants showed a slight increase in bond stability to collagen type III. This effect probably arises from additional hydrogen bonds that come along with the introduction of these mutations. Using the same mutants, but collagen type VI as a binding partner, resulted in a significant increase in bond stability. VWF domain A1 was reported to be essential for the interaction with collagen type VI and thus our findings strengthen the hypothesis that the VWF A1 domain can compensate for mutations in the VWF A3 domain. Additionally, our data suggest that the mutations could even stabilize the interaction between VWF and collagen without shear. VWF-collagen interactions seem to be an important system in which defective interactions between one VWF domain and one type of collagen can be compensated by alternative binding events.

Mutual A domain interactions in the force sensing protein von Willebrand factor.

The von Willebrand factor (VWF) is a glycoprotein in the blood that plays a central role in hemostasis. Among other functions, VWF is responsible for platelet adhesion at sites of injury via its A1 domain. Its adjacent VWF domain A2 exposes a cleavage site under shear to degrade long VWF fibers in order to prevent thrombosis. Recently, it has been shown that VWF A1/A2 interactions inhibit the binding of platelets to VWF domain A1 in a force-dependent manner prior to A2 cleavage. However, whether and how this interaction also takes place in longer VWF fragments as well as the strength of this interaction in the light of typical elongation forces imposed by the shear flow of blood remained elusive. Here, we addressed these questions by using single molecule force spectroscopy (SMFS), Brownian dynamics (BD), and molecular dynamics (MD) simulations. Our SMFS measurements demonstrate that the A2 domain has the ability to bind not only to single A1 domains but also to VWF A1A2 fragments. SMFS experiments of a mutant [A2] domain, containing a disulfide bond which stabilizes the domain against unfolding, enhanced A1 binding. This observation suggests that the mutant adopts a more stable conformation for binding to A1. We found intermolecular A1/A2 interactions to be preferred over intramolecular A1/A2 interactions. Our data are also consistent with the existence of two cooperatively acting binding sites for A2 in the A1 domain. Our SMFS measurements revealed a slip-bond behavior for the A1/A2 interaction and their lifetimes were estimated for forces acting on VWF multimers at physiological shear rates using BD simulations. Complementary fitting of AFM rupture forces in the MD simulation range adequately reproduced the force response of the A1/A2 complex spanning a wide range of loading rates. In conclusion, we here characterized the auto-inhibitory mechanism of the intramolecular A1/A2 bond as a shear dependent safeguard of VWF, which prevents the interaction of VWF with platelets.

Characterizing the effect of polymyxin B antibiotics to lipopolysaccharide on Escherichia coli surface using atomic force microscopy.

Lipopolysaccharide (LPS) on gram-negative bacterial outer membranes is the first target for antimicrobial agents, due to their spatial proximity to outer environments of microorganisms. To develop antibacterial compounds with high specificity for LPS binding, the understanding of the molecular nature and their mode of recognition is of key importance. In this study, atomic force microscopy (AFM) and single molecular force spectroscopy were used to characterize the effects of antibiotic polymyxin B (PMB) to the bacterial membrane at the nanoscale. Isolated LPS layer and the intact bacterial membrane were examined with respect to morphological changes at different concentrations of PMB. Our results revealed that 3 hours of 10 µg/mL of PMB exposure caused the highest roughness changes on intact bacterial surfaces, arising from the direct binding of PMB to LPS on the bacterial membrane. Single molecular force spectroscopy was used to probe specific interaction forces between the isolated LPS layer and PMB coupled to the AFM tip. A short range interaction regime mediated by electrostatic forces was visible. Unbinding forces between isolated LPS and PMB were about 30 pN at a retraction velocity of 500 nm/s. We further investigated the effects of the polycationic peptide PMB on bacterial outer membranes and monitored its influences on the deterioration of the bacterial membrane structure. Polymyxin B binding led to rougher appearances and wrinkles on the outer membranes surface, which may finally lead to lethal membrane damage of bacteria. Our studies indicate the potential of AFM for applications in pathogen recognition and nano-resolution approaches in microbiology.

Detailed Evidence for an Unparalleled Interaction Mode between Calmodulin and Orai Proteins.

Calmodulin (CaM) binds most of its targets by wrapping around an amphipathic α-helix. The N-terminus of Orai proteins contains a conserved CaM-binding segment but the binding mechanism has been only partially characterized. Here, microscale thermophoresis (MST), surface plasmon resonance (SPR), and atomic force microscopy (AFM) were employed to study the binding equilibria, the kinetics, and the single-molecule interaction forces involved in the binding of CaM to the conserved helical segments of Orai1 and Orai3. The results consistently indicated stepwise binding of two separate target peptides to the two lobes of CaM. An unparalleled high affinity was found when two Orai peptides were dimerized or immobilized at high lateral density, thereby mimicking the close proximity of the N-termini in native Orai oligomers. The analogous experiments with smooth muscle myosin light chain kinase (smMLCK) showed only the expected 1:1 binding, confirming the validity of our methods.

Calibrated complex impedance of CHO cells and E. coli bacteria at GHz frequencies using scanning microwave microscopy.

The application of scanning microwave microscopy (SMM) to extract calibrated electrical properties of cells and bacteria in air is presented. From the S 11 images, after calibration, complex impedance and admittance images of Chinese hamster ovary cells and E. coli bacteria deposited on a silicon substrate have been obtained. The broadband capabilities of SMM have been used to characterize the bio-samples between 2 GHz and 20 GHz. The resulting calibrated cell and bacteria admittance at 19 GHz were Y cell = 185 µS + j285 µS and Y bacteria = 3 µS + j20 µS, respectively. A combined circuitry-3D finite element method EMPro model has been developed and used to investigate the frequency response of the complex impedance and admittance of the SMM setup. Based on a proposed parallel resistance-capacitance model, the equivalent conductance and parallel capacitance of the cells and bacteria were obtained from the SMM images. The influence of humidity and frequency on the cell conductance was experimentally studied. To compare the cell conductance with bulk water properties, we measured the imaginary part of the bulk water loss with a dielectric probe kit in the same frequency range resulting in a high level of agreement.

High-speed AFM images of thermal motion provide stiffness map of interfacial membrane protein moieties.

The flexibilities of extracellular loops determine ligand binding and activation of membrane receptors. Arising from fluctuations in inter- and intraproteinaceous interactions, flexibility manifests in thermal motion. Here we demonstrate that quantitative flexibility values can be extracted from directly imaging the thermal motion of membrane protein moieties using high-speed atomic force microscopy (HS-AFM). Stiffness maps of the main periplasmic loops of single reconstituted water channels (AqpZ, GlpF) revealed the spatial and temporal organization of loop-stabilizing intraproteinaceous H-bonds and salt bridges.

Nanopharmacological Force Sensing to Reveal Allosteric Coupling in Transporter Binding Sites.

Controversy regarding the number and function of ligand binding sites in neurotransmitter/sodium symporters arose from conflicting data in crystal structures and molecular pharmacology. Here, we have designed novel tools for atomic force microscopy that directly measure the interaction forces between the serotonin transporter (SERT) and the S- and R-enantiomers of citalopram on the single molecule level. This approach is based on force spectroscopy, which allows for the extraction of dynamic information under physiological conditions thus inaccessible via X-ray crystallography. Two distinct populations of characteristic binding strengths of citalopram to SERT were revealed in Na(+)-containing buffer. In contrast, in Li(+) -containing buffer, SERT showed only low force interactions. Conversely, the vestibular mutant SERT-G402H merely displayed the high force population. These observations provide physical evidence for the existence of two binding sites in SERT when accessed in a physiological context. Competition experiments revealed that these two sites are allosterically coupled and exert reciprocal modulation.